Early in bone development, chondrocytes and cells of the osteoblast lineage proliferate and differentiate in a regulated fashion. Parathyroid hormone-related protein (PTHrP), a key modulator of these processes, signals through a G protein-coupled receptor, the PTH/PTHrP receptor (PPR) to delay hypertrophic differentiation of chondrocytes in fetal life. In preliminary studies we have found that in postnatal life, the removal of the PPR leads quickly to the disappearance of the growth plate (growth plate fusion). In Aim 1 we will confirm these findings, use genetic tools to determine the roles of specific G proteins in mediating the postnatal actions of the PPR, and determine the mechanism of growth plate fusion in this model. In Aim 2, we will continue to determine the ways that chondrocyte hypertrophy is regulated. In the first sub-aim we will use genetic tools both in vivo and in bone explants to test the hypothesis that PTHrP regulates hypertrophy by stopping the interaction of HDAC4 with MEF2c. In the second sub-aim, we will study the role of a novel transcriptional regulator, limb bud and heart (LBH) in regulating chondrocyte hypertrophy. Our studies in fetal chick limbs have demonstrated that LBH regulates both chondrocyte and osteoblast development by suppressing expression of the transcription factor, Runx2. Here we will use the cre-lox approach to knock LBH out of chondrocytes (collagen 11 promoter) and osteoblasts (osterix promoter) to determine to roles of LBH in bone development. We will verify in the mouse knockout model that LBH regulates hypertrophy and explore possible connections between the actions of LBH and the PTHrP-HDAC4-MEF2c axis. In the previous grant cycle, we used tamoxifen-regulated osterix-cre and collagen l-cre constructs to trace the osteoblast lineage in early bone development. In Aim 3, we will focus on earlier steps of the osteoblast lineage by generating further tamixofen-regulated cre lines driven by the Msx2 and Runx2 promoters. These mice will be used to assess the roles and regulation of very early cells of the osteoblast llneage.assess the roles and regulation of very early cells of the osteoblast lineage..